We are proposing this project to develop and commercialize a novel, histidine/lysine (HK) polymer based, siRNA transfection reagent. The goal of the phase I study of this project is to evaluate the feasibility of using PT88, a member of the HK polymer family, as siRNA transfection reagent. RNA interference (RNAi) is a process of sequence-specific gene silencing mediated by double-stranded RNA-molecules. In mammalian cells, the phenomenon can be invoked by introducing small 19-21 nucleotide long RNA duplexes, known as small interfering RNA (siRNA) into cells. Due to the high potency of siRNA, the use of siRNA to selectively silence gene expression at mRNA levels has rapidly replaced antisense and ribozyme in cell culture and animal models. Currently, there are several commercial available reagents for siRNA transfection that include liposome-based transfection reagents and polymer-based transfection reagents. In addition to its toxicity to the cells, the liposome-based transfection reagents are very sensitive to serum in the cell culture medium, thus require a complicated transfection procedure and are not suitable for high through- put studies. On the other hand, currently available polymer-based transfection reagents are highly toxic to the cells, and its toxicity to the host cells may well shadow the effect of siRNA-mediated gene silencing. Therefore, a high efficient, low toxic siRNA transfection reagent is highly desired and its commercialization will significantly advance the application of siRNA in the field of gene function study and development of new therapeutics. We have developed a group of HK polymers and utilized them as nucleic acid carrier for in vitro and in vivo delivery. The in vitro transfection efficiency of plasmid DNA mediated by the HK polymers is comparable to that of polymer-based transfection reagent, while the HK polymers give a much higher in vivo transfection efficiency than both polymer-based and liposome-based transfection reagents. More importantly, the HK polymers present strong serum tolerances and exhibit minimum, if any, cytotoxicity to the transfected cells. We found that PT88, a member of the HK polymers, demonstrates a great capability of mediating siRNA transfection and siRNA/plasmid DNA co-transfection in culture cells. This phase I project is therefore proposed to evaluate the feasibility of PT88 as a commercial siRNA transfection reagent. The specific aims of Phase I include 1) evaluate the efficiencies of PT88 mediated siRNA transfection and siRNA/DNA co-transfection in 10 cell lines that include cell lines believed easy to be transfected and cell lines believed difficult to be transfected, 2) perform comprehensive cytotoxicity study and serum tolerances study of PT88 in the cultured cells, and 3) investigate the feasibility of PT88 for high through- put siRNA transfection that include multiple transfection and co-transfection of plasmid DNA and siRNA duplexes. The success of the Phase I will lead to a Phase II project that focuses on commercial development of the PT88 HK polymer as a siRNA transfection and siRNA/DNA co-transfection reagent. The annual market value for a dominant siRNA transfection reagent is easily over tens of millions of dollars, giving the huge potentials of siRNA as a tool for biomedical research as well as a therapeutic modality.